Analysis of the juxtamembrane dileucine motif in the insulin receptor

Dileucine-containing motifs are involved in trans-Golgi sorting, lysosomal targeting, and internalization. Previously, we have shown that the dileucine motif (EKITLL, residues 982-987) in the juxtamembrane region of the insulin receptor is involved in receptor internalization. Substitution of alanine residues for Leu(986) and Leu(987) led to a 3- to 5-fold decrease in the ability of the receptors to mediate insulin uptake. In the current study, we show that mutation of the same motif to Met(986)Ser(987), the sequence found in the homologous position in the type I insulin-like growth factor receptor, did not affect insulin uptake. Therefore, we inquired whether the sequence EKITMS as an isolated motif could mediate the targeting of a reporter molecule to endosomes and then lysosomes, as was shown previously with the EKITLL motif of the normal receptor. Chimeric molecules containing Tac antigen fused to different hexapeptide sequences showed distinct patterns of subcellular localization by immunofluorescence microscopy. Tac-EKITLL and Tac-EKITAA were found predominantly in lysosomes and the plasma membrane, respectively. In contrast, Tac-EKITMS was found at the plasma membrane, in the trans-Golgi network, and in endosomes, but only small amounts were found in lysosomes. Thus, the dileucine motif (EKITLL) plays an important role in directing endocytosis of the intact insulin receptor and in mediating efficient endocytosis and lysosomal targeting as an isolated motif. Substitution of AA for LL inhibits endocytosis and lysosomal targeting in both systems. In contrast, substitution of MS for LL permits rapid endocytosis in the intact receptor, but mediates modest endocytosis and very little targeting to lysosomes as an isolated motif. Our observations support the idea that sorting signals are recognized at multiple steps in the cell, and that specific amino acid substitutions may differentially affect each of these sorting steps.